PROJECT SUMMARY/ABSTRACT Multidrug resistant (MDR) infections caused by the bacterial pathogen Acinetobacter baumannii (Ab) are increasing at alarming rates. Today the MDR frequencies among Ab clinical isolates are higher than any other Gram-negative bacterium. For this reason, the World Health Organization has categorized Ab as top priority for the research and development of new antimicrobial therapies. Ab is largely associated with healthcare- acquired infections, namely pneumonia and septicemia. However, the ability of Ab to cause urinary tract infections (UTI), including catheter-associated UTI (CAUTI), is underappreciated. Despite that 20-30% of the Ab isolates come from urinary sources, there is no established model to study Ab UTI or the molecular basis of uro-pathogenic Ab (UPAb) virulence. As UPAb are contributing to the rise of MDR UTI globally, there is a pressing need to create new Ab UTI therapies. Thus, there is demand for a murine model to study MDR-UPAb and to identify the bacterial factors critical for Ab UTI. We have developed the first Ab catheter-associated UTI (CAUTI) murine model to examine implant and bladder colonization in mice, and we have selected the MDR Ab isolate AbCA1 as model strain. AbCA1 was isolated from a female UTI patient in Argentina. The strain belongs to the international Ab clone ST25, a phylogenetically distinct and emergently important lineage of Ab clinical strains whose pathophysiology has been poorly studied. AbCA1 carries a large MDR-encoding plasmid, pAB5, which belongs to a family of Ab Large Conjugative Plasmids (AbLCPs) identified in multiple, geographically-diverse Ab strains. Interestingly, our preliminary findings show that pAB5 confers improved survival in our CAUTI model, but reduced virulence in a pneumonia model. Adherence to the catheter and urothelium constitutes the first stage in establishing CAUTI. We have validated our model showing that deletion of chaperone-usher pili abrogates AbCA1 ability to colonize the catheter and bladder in our CAUTI model. Similarly, deletion of the glycoprotease CpaA, one of the most abundantly expressed T2SS-associated effector, diminished AbCA1 growth in human urine in vitro and virulence in the CAUTI model. In this application, we propose to use our recognized expertise in Ab molecular biology and pathogenesis to investigate the bacterial factors involved in the early stages of Ab CAUTI. We will further investigate the factors involved in adherence and the extracellular and surface-exposed elements involved in UPAb pathogenesis. We will also investigate how pAB5 enhances the uro-pathogenic state of AbCA1. Our work reveals that Ab is not a homogenous group of pathogens with a stagnant battery of virulence factors; instead, strains appear to acquire unique traits that better equip them to cause disease in specific host niches. Establishing a model Ab strain and an animal disease model that represents these neglected infections is critical to efforts to stop UPAb from contributing to the growing global burden of MDR UTI. Our proposed models and investigations will serve as foundation for the development of novel diagnostics and therapeutics.